Several selenium-containing proteins of bacterial origin are being investigated. Thiolase and 3-hydroxybutyryl-CoA dehydrogenase from Clostridium kluyveri contain selenium as selenomethionine. Detailed studies have been conducted on the localization and distribution of selenium throughout the primary structure of these enzymes. Peptide mapping experiments using reverse phase HPLC were performed with the purified enzymes labelled with 75Se and 35S. The results of these studies indicated that selenium was distributed throughout the primary structure of thiolase and 3-hydroxybutyryl-CoA dehydrogenase in a manner that paralleled methionine. Analyses of proteolytic digests of these proteins, support a nonspecific incorporation mechanism of selenium. However, this mechanism appears to be limited to selenomethionine substitution for methionine since no selenium incorporation as selenocysteine was detected. Selenoprotein A of the clostridial glycine reductase complex which contains selenium as selenocysteine is also being investigated. An improved method for the purification of selenoprotein A from both Clostridium sticklandii and Clostridium purinolyticum has been developed using an ion exchange HPLC step. Milligram quantities of this protein have now become available for structural studies. In constrast to thiolase and 3-hydroxybutyryl-CoA dehydrogenase, selenium has been found to be localized at a specific residue within the polypeptide chain. A tryptic peptide has been isolated by reverse phase HPLC which contains the selenocysteine residue. The peptide has been partially sequenced and also appears to contain the two cysteine residues of the protein. The selenocysteine residue however, occurs in a unique position near the carboxylterminus of the protein as shown with carboxypeptidase Y digestion.